The Role of Late MMR Proteins in DNA Repair and Cancer

描述（由申请人提供）：我们研究的主要目标是分析哺乳动物DNA错配修复系统（MMR）的生物学功能，并确定MMR基因突变如何影响DNA修复和癌症易感性。MMR对于维持哺乳动物基因组的完整性是必不可少的，并且MMR基因中的突变导致癌症易感性增加和减数分裂失败。真核MMR是一个复杂的系统，需要几个MutS和MutL蛋白的相互作用来启动修复反应。在错配识别之后，下游事件被激活，导致错误掺入的核苷酸的切除和通过DNA再合成填充所得的单链缺口。在过去的资助期内，我们对核酸外切酶1（Exol）突变小鼠进行了全面分析，这是目前已知在真核切除反应中起作用的唯一一种核酸外切酶。我们确定Exol在Msh 2-Msh 6依赖性碱基-碱基错配修复中起作用，并且Exol失活导致高度渗透的癌症易感性表型。此外，Exol功能的丧失导致雄性和雌性小鼠的不育，表明Exol在哺乳动物减数分裂中的重要作用。我们假设，一组靶向错义突变的产生将阐明Exol如何发挥作用来抑制癌症，并将允许研究其在减数分裂中的作用。此外，鉴定与Exol相互作用的蛋白质的努力将有助于阐明MMR晚期的其他关键组分。该提案的具体目标是：1.对人类癌症患者中发现的人类Exol错义突变以及小鼠中普遍存在的编码单核苷酸多态性（SNP）进行建模，并分析所得癌症易感性表型。2.确定Exol错义突变对MMR和突变避免的影响。我们将分析Exol敲入突变对体外DNA修复功能的影响，并确定小鼠组织中产生的体内突变体表型。3.确定错义突变对Exol在哺乳动物减数分裂中的生物学功能的影响。我们将进行详细的组织病理学和细胞遗传学研究，以确定Exol错义突变可能破坏精母细胞和卵母细胞中的I期前期进展的机制。4.分析一种新的Exol相互作用蛋白在MMR中的意义，并建立一种MMR复合物的体内分析系统。我们已经确定RuvBL 2解旋酶作为Exol相互作用酶，并将研究其在MMR中的潜在作用。我们还建议建立一个在体内系统的MMR复合物的形成分析和新的MMR相关蛋白在小鼠组织中的鉴定。

DESCRIPTION (provided by applicant): The main goal of our research is to analyze the biological functions of the mammalian DNA mismatch repair system (MMR) and to determine how mutations in MMR genes affect DNA repair and cancer susceptibility. MMR is essential for maintaining the integrity of the mammalian genome and mutations in MMR genes result in increased cancer susceptibility and meiotic failure. Eukaryotic MMR is a complex system that requires the interaction of several MutS and MutL proteins for the initiation of the repair reaction. Subsequent to mismatch recognition, downstream events are activated that lead to the excision of the misincorporated nucleotide(s) and the filling in of the resulting single strand gap by DNA resynthesis. In the past funding period, we performed a comprehensive analysis of Exonuclease 1 (Exol) mutant mice, the only currently identified exonuclease known to function in the eukaryotic excision reaction. We determined that Exol functions in the Msh2-Msh6-dependent repair of base-base mismatches and that Exol inactivation causes a highly penetrant cancer predisposition phenotype. In addition, loss of Exol function caused infertility in male and female mice, indicating an essential role for Exol in mammalian meiosis. We hypothesize that the generation of a set of targeted missense mutations will elucidate how Exol functions to suppress cancer and will allow a study of its role in meiosis. In addition, efforts to identify proteins that interact with Exol, will help elucidate other key components of the late stages of MMR. The specific aims of this proposal are: 1. To model human Exol missense mutations found in human cancer patients as well as prevalent coding single nucleotide polymorphisms (SNPs) in mice and analyze the resulting cancer susceptibility phenotype. 2. To determine the effect of Exol missense mutations on MMR and mutation avoidance. We will analyze the effect of the Exol knock-in mutations on DNA repair functions in vitro and determine the resulting in vivo mutator phenotype in mouse tissues. 3. To determine the effects of missense mutations on the biological functions of Exol in mammalian meiosis. We will perform detailed histopathological and cytogenetic studies to determine the mechanisms by which Exol missense mutations may disrupt prophase I progression in spermatocytes and oocytes. 4. To analyze the significance of a novel Exol interacting protein for MMR and to establish a system for the in vivo analysis of MMR complexes. We have identified RuvBL2 helicase as an Exol interacting enzyme and will study its potential role in MMR. We also propose to establish an in vivo system for the analysis of MMR complex formation and the identification of novel MMR associated proteins in mouse tissue.